The aims of this research were to develop and an aequorin-based approach for measuring cytosolic Ca<sup>2+</sup> ([Ca<sup>2+</sup>]<sub>c</sub>) in living hyphae of <i>Neurospora crassa </i>and to use this method to investigate the contribution of individual proteins to the generation of the specific Ca<sup>2+</sup>-signatures associated with [Ca<sup>2+</sup>]<sub>c</sub> transients. Molecular and genomic methods were also used to identify Ca<sup>2+</sup>-signalling proteins in <i>Neurospora crass, Aspergillus fumigatus </i>and <i>Magnaporthe grisea.</i> Results confirmed that a reliable method for the quantitative measurement of [Ca2+]c in living <i>N. crassa</i> hyphae had been developed with the aequorin reporter system. This method was used to characterise Ca<sup>2+</sup>-signatures in <i>N. crassa </i>in response to (a) mechanical perturbation, (b) hypo-osmotic shock and (c) high external Ca<sup>2+</sup> under different environmental conditions. Ca<sup>2+</sup>-signatures in response to these stimuli were shown to have a unique set of characteristics in response to each stimulus. These characteristics were apparent under all the conditions tested. Ca<sup>2+</sup>-signatures in response to the three stimuli were measured in wild-type <i>N. crassa</i> treated with Ca<sup>2+</sup> antagonists and agonists and in untreated mutant strains of <i>N. crassa</i> compromised in Ca<sup>2+</sup>-signalling. In each case, differences in Ca<sup>2+</sup>-signatures could be quantitatively measured. Cloning of the <i>cot-4</i> gene in the <i>cot-4</i> morphological mutant of <i>N. crassa</i> showed it to encode the catalytic subunit of calcineurin, a Ca<sup>2+</sup>/calmodulin dependent protein phosphatase. An analysis of the genomes of <i>N. crassa</i>, <i>A. fumigatus </i>and <i>M. grisea</i> identified for the first time, many of the key Ca<sup>2+</sup>-signalling proteins present in filamentous fungi. An inventory of Ca<sup>2+</sup>-signalling proteins in filamentous fungi is an important starting point for reverse genetic and physiological approaches aiming at elucidating the biological significance of these proteins.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:664218 |
| Date | January 2004 |
| Creators | Zelter, Alexander |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13249 |
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